*2.3. Outcomes*

Clinical and serological outcomes were considered in the study. The clinical outcomes included an IBS-SSS score decrease of ≥50 points, reduction of at least 30% in abdominal pain [33] and bloating by a visual analogic scale (VAS), normalization of stool shape by Bristol Stool Scale and IBS-QoL overall score increase ≥14 points [34,35]. The serological outcomes included the variation in inflammatory cytokines and zonulin levels during the follow-up period to assess potential changing of immune-inflammatory activity and of intestinal permeability, respectively. Both clinical and serological outcomes were evaluated in the overall study population and according to treatment duration.

### *2.4. Statistical Analysis*

Continuous variables were expressed as median and 95% confidence interval (CI) or as mean ± standard deviation (SD). The normality of data distribution was tested by D'Agostino-Pearson test. The comparison between quantitative variables was carried out using the Student *t*-test; for the

comparison between values at T0 and at T1, the Student *t*-test for paired measurements was used. The comparison between qualitative variables was carried out by Fisher exact test or chi-squared test for trend where appropriate. Nonparametric variables were analysed with the Wilcoxon test for paired samples and with the Mann-Whitney test for independent samples. The correlation between quantitative variables was carried out by non-parametric Spearman test. The results of all analyses were considered significant for *p* values < 0.05. All statistical analyses were performed using MedCalc software version 18.9.1 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org; 2018).
